Cytoplasmic dynein is a microtubule-based motor with critical and diverse cellular functions, ranging from vesicle transport to mitotic spindle with a distinctive structure, is a required co-factor for most o the ese functions of dynein. However, it is still not known why dynactin is required dt activate cytoplasmic dynein function. To address these questions, the studies proposed have three specific aims. In specific aim I, we will examine the mechanochemical coupling of cytoplasmic dynein, and the effects of dynactin on this mechanochemistry: does dynactin significantly alter the parameters of the microtubule-dynein ATPase cycle? While evidence suggests that dynactin functions as a vesicle-bound receptor for dynein, it may also be involved more actively in constraining aspects of motility. This hypothesis can be tested by analyzing the dynein ATPase cycle, and the effects of dynactin on this pathway. In specific aim II, we will examine the coupling mechanisms linking dynein and dynactin to cellular cargo: are there dynactin-dependent as well as dynactin-independent mechanisms of cargo-coupling in the cell? We will test the hypothesis that dynein couples to cargo through multiple and distinct mechanisms, which may provide specificity of targeting and allow for very specific regulation of function. And in specific aim III, we will examine the role of dynein/dynactin localization to dynamic microtubule ends: is there a specific role for dynein and dynactin in a microtubule plus-end complex involved in microtubule search-and-capture? Do specific associations of dynein and dynactin with EB 1 and CLIP- 170 at microtubule plus ends mediate interactions of microtubules with cellular organelles or with the cortex? The interaction between dynein and dynactin appears to be conserved from yeast to humans. This conservation may be due to a fundamental need for dynactin in adapting dynein to intracellular transport functions. However, we have much to learn about the complex interactions of dynein and dynactin which are required to produce cargo motility along microtubules, and which are required to target these multifunctional proteins specifically and effectively within the cell. Successful completion of the studies proposed should significantly improve our understanding of how dynein and dynactin function in essential cellular processes, such as eukaryotic cell division and vesicle trafficking. Therefore, this work will provide further understanding of the processes which become disrupted in disease, such as the uncontrolled cell division seen in cancer, and the disrupted axonal transport that has been linked to neurodegenerative diseases such as ALS.